The present invention refers to the use of novel molecules able to bind tenaciously to silica, borosilicate and silicate surfaces, and thus to modify their properties and characteristics. When applied to silica-based chromatography, it offers important advantages in all cases in which it is necessary to modulate the interaction of analytes with the stationary phase. In capillary zone electrophoresis (CZE), such compounds will eliminate or invert the electroendoosmotic (EEO) flow, greatly simplifying the analysis of negatively-charged compounds and permitting the analysis of bio(macro)molecules via the direct use of naked capillaries.
The fused silica is constituted of three types of ionizable silanols: isolated, geminal and vicinal. The density of such groups has been estimated of the order of 5 silanols per nm2, whose average pKa, value has been estimated as 6.3 (M. S. Bello, L. Capelli e P. G. Righetti, J. Chromatogr. A 684, 1994, 311). Thus, at any operative pH value above 2, there will be a fraction of ionized silanols, fraction which will be larger and larger at progressively higher pH values till reaching a plateau at pH ca. 10.
The EEO flow in a fused silica column is produced by the electric field and is transmitted by the drag of ions in a thin liquid layer adjacent to the silica wall. The origin of the net positive charge in this thin liquid sheath is due to the progressive ionization of silanol groups in the wall. The electric potential generated by these fixed negative charges generates a diffuse double layer (called Debye-Hxc3xcckel layer) in which there exist an excess of cations as compared with anions in the buffer present in solution. When the electric circuit is closed, this excess of cations is continuously perturbed and drugged toward the negative pole (the cathode). Since the cations coordinate a number of hydration-water molecules, the continuous migration of this excess of cations generates a net water transport from the anode to the cathode, called EEO flow. This flux continues as long as the electric field is applied, since the Debye-Hxc3xcckel double layer is continuously perturbed by the applied potential difference and thus it has to be continuously reformed. The EEO flow in CZE has been studied in depth, due to its fundamental importance in understanding the results of electrophoretic separations e due to its strong influence on the reproducibility of transit times. The reproducibility of the EEO flow is in fact rather modest, particularly in proximity of the pKa, where the EEO vs. pH curve exhibits the highest slope. This is also due to the slow equilibration of the silica surface in changing from acidic to alkaline solutions, due, for instance, to strongly acidic or basic pH values adopted in washing the silica column after electrophoretic analysis of complex analytes, which could leave material adhering to the wall. This slow equilibration process causes dramatic variations of the EEO flux, which, in turn, could provoke poor reproducibility of the transit times of analytes, both between runs and during different days of analysis.
Per se, the EEO flux is not noxious to the electrophoretic process; on the contrary its presence is of fundamental importance when attempting separation in a single run of mixtures of anionic, cationic and neutral substances. At elevated EEO fluxes, it is possible that even negatively charged analytes, which would normally migrate to the anode, will be transported to the cathode, thus being detected at the monitoring window (in normal polarity runs the cathode is placed close to the detector). The presence of the EEO flux is of fundamental importance in methods such as electrokinetic micellar chromatography (MEKC), in which the analytes are adsorbed onto a surfactant (typically Na dodecyl sulphate, SDS). Since the surfactant micelles migrate towards the anode, but generally with lower velocities as compared with that of the EEO flux, at appropriate pH values, there is a large time window for separating both neutral and hydrophobic analytes which interact to some extent with said micelles. On the contrary, in numerous other cases, the presence of negative charges on the wall (to which the EEO flux is associated) is strongly detrimental to the electrophoretic separation. One of the most serious problems, in this case, is the adsorption of cationic analytes. Whereas such adsorption, in the case of small molecules, might be of modest entity, reversible and thus provoke only moderate losses of resolution, in the case of macromolecules, especially for proteins and peptides, this phenomenon is disastrous and could cause not only strong peak asymmetry, but even complete loss of analyte when totally and irreversibly adsorbed to the wall. Even in the case of DNA separations such EEO flow is noxious, since it causes peak asymmetry and elution of sieving liquid polymers from the capillary lumen. Over the years, many solutions have been proposed for solving this problem as reviewed in e.g., M. Chiari, M. Nesi e P. G. Righetti, in Capillary Electrophoresis in Analytical Biotechnology, P. G. Righetti, Ed., CRC Press, Boca Raton, 1996, pp. 1-36; F. E. Regnier e S. Lin, in High Performance Capillary Electrophoresis, M. G. Khaledi, Ed., Wiley, New York, 1998, pp. 683-728; G. M. McLaughilin et K. W. Anderson, in High Performance Capillary Electrophoresis, M. G. Khaledi, Ed., Wiley, New York, 1998, pp. 637-681.
Among the various solutions proposed for eliminating the EEO flux, we can recall here:
a) Variations in the type of buffer and its additives;
b) Adsorbed coatings (e.g., neutral polymers, neutral, charged or zwitterionic surfactants);
c) Covalently bound polymers, typically neutral macromolecules, such as acrylamides and celluloses, bound to the wall usually via bifunctional molecules (bridging or cross linking agents).
Covalently bound polymers have been found to be the most effective in quenching EEO flux, not only because the wall should be physically carpeted with neutral polymers, but also because, due to the anchoring of the polymers to the free silanols, there is an overall suppression of negative charges. However, such coatings are the most expensive among those offered on the market, and cannot be easily performed in individual laboratories, since good skills in organic chemistry and specialized equipment are required. In addition, this type of coating undergoes progressive deterioration during use, which calls for replacement of the capillary, this adding to the costs of analysis.
For all these reasons, dynamic capillary coatings, as obtained by additives to the background electrolyte, have been much preferred and definitely more popular among users. Among the buffer modifications there could be very simple ones, such as changes of the operative pH (e.g., at pH extremes the proteins are either repelled by the capillary, at alkaline pHs, or are not adsorbed, because the wall is neutral, at acidic pHs), or changes in the type of cation, or even the use of hydro-organic solvents, or yet strong changes in the buffer molarity (at high buffer concentrations interactions with the capillary wall are quenched or discouraged).
Each of these modifications can present some advantages, but also a number of disadvantages. A highly promising research line is the one which utilizes oligo-amines (especially tri-, tetra- and penta-amines). Oligo-amines are adsorbed to the wall via cooperative linkages, due to the presence of multiple charges on the skeleton of nitrogens and are thus able to minimize and often complete eliminate protein and peptide adsorption to the wall. Among these classes of compounds, the best ones appear to be spermine (a skeleton of four nitrogens separated by two or three carbon atoms) and TEPA (tetraethylene penta-amine) composed by a skeleton of five nitrogens separated by ethylene groups. This last molecule belongs to a large family of polyazotated compounds, both linear and branched. It would appear that the efficacy of such oligo-aminic compounds increases as a function of molecular mass as well as of the CH2/NH ratio and of the total number of ethylene groups in the molecule.
Even though the oligo-amines appear to be extremely promising both because of the ease of their use and for the efficiency of the coating, they present a common, most prominent defect: at neutral and alkaline operative pH values (the latter being the most popular for protein separations) they exhibit a drastic decrease of efficacy, since their nitrogens are progressively deprotonated, this in turn hampering the co-operative linkage to the wall (such linkage being mostly of ionic type).
Also in chromatographic processes utilizing silica beads as supports for covalent linkage of a variety of phases, silanol ionization represents a serious problem. For instance, in reversed-phase (RP)-HPLC, many companies produce silica spheres, to which hydrophobic phases, such a C18H37 (C18 phases) or C8H17 (C8 phases), are covalently affixed. Although reactions are carried out under conditions which should ensure full reaction of free silanols, in practice, due to steric hindrance, barely 50% of the silanol population can react (J. C. Dolan, Liquid Chrom. Gas Chrom. Int. 12, 1999, 156; D. V. McCalley, Liquid Chrom. Gas Chrom. Int. 12, 1999, 638; D. C. Leach, M. A. Stadalius, J. S. Berus and L. R. Snyder Liquid Chrom. Gas Chrom. Int. 1, 1988, 22). As a consequence, in the separation of basic compounds, peaks are strongly tailed with loss of resolution, and sometimes even total loss of analyte occurs, due to irreversible adsorption onto the silanolic phase. As a remedy, one has tried to react free silanols (the ones still not bound with C18, C8 phases etc.) with silanic agents of small size, such as trimethylchloro silane, a procedure called end capping. However, even end-capped phases still present xc2xd of the silanols unreacted, which means that the problems is lessened but not abolished. In order to further minimize this problem, in silica-based chromatography, already in the seventies, additives to the eluent have been proposed, able to block ionized silanols via salt bridges. Among those additives, the most popular one is triethylamine, at concentration 20-50 mM. The compounds described in the present invention, being able to bind to free silanols, are highly efficient in ameliorating chromatographic separation, as described below.
The present invention describes a novel class of molecules able to overcome all the drawbacks described above.
The compounds here claimed possess the following structural characteristics:
a) the presence of one or more quaternary nitrogens able to form ionic bonds with silanols at any operative pH value;
b) the presence of one or more basic atoms (tertiary nitrogen or oxygen, either ethereal or carbonyl) able to form hydrogen bonds or electrostatic interactions via the same heteroatoms at different pH values along the pH scale;
c) the presence of one or more alkyl chains (typically but not exclusively C-4), possessing terminal carbon atoms substituted with one or more electronegative atoms able to react with silanolic groups to such an extent as to form covalent bonds with the capillary wall.
Particularly effective appear to be quaternary ammonium salts derivatives possessing the structural formula 1, 2 and 3. 
where R represents typically (but not exclusively) a CH3, whereas Rxe2x80x2 and Rxe2x80x3, independently-between them, represent typically (but not exclusively) either a CH3, or a group with formula xe2x80x94[(CH2)n]xe2x80x94Z, where n=2o greater than 2, preferably 4, and Z=halogen, OH, O-alkyl (with 1-4 carbon atoms), Oxe2x80x94SO2C6H5CH3, N3. Compounds of relevant interest are also the heterocyclic derivatives of formula 3, where X=O, Yxe2x89xa0; or X=C, Y=O; or X=CH, Y=ORxe2x80x2xe2x80x3; or X=CH, Y=H, alkyl (C1-C10).
The preferred substituents in compounds of formula 1, 2 and 3 are R=CH3, Rxe2x80x2=CH3, Rxe2x80x3=(CH2)4I.
Other compounds covered by the present invention are molecules of type above indicated where the heterocyclic ring and/or the alkyl residues contain one or more asymmetric carbons so as to be utilized as chiral selectors.
The compounds described in the present invention can be utilized either as additives to the background electrolyte (typically at acidic pH values), or as wall modifiers introduced only during the pre-conditioning phase of the capillary (typically at neutral or alkaline pH values). Under the latter conditions, given the absence of the modifier in the background electrolyte, one obtains the unique advantage of ameliorating the signal to noise ratio, thus improving post-column techniques for analyte detection.